Fig. 4. IP₃R-mediated Ca⁺⁺ release is required for the initial IL-2 and IFNγ gene expression.

(A) CD4 T cells were activated with anti-CD3 and anti-CD28 mAbs in medium alone or in the presence (APB) of 2-APB (15 μM) and IL-2 production was examined by ELISA on day 2. (B) CD4 T cells were activated with anti-CD3/anti-CD28 mAbs and after two days medium or 2-APB was added to the culture. IL-2 production was examined by ELISA two days later (day 4). Results are representative of at least three experiments. (C) CD4 T cells were activated with anti-CD3 and anti-CD28 mAbs in the presence of DMSO as a vehicle (Veh) or Xe-C (5 μM) and after two days IL-2 and IFNγ production were examined by ELISA. (D) CD4 T cells were activated with anti-CD3 and anti-CD28 mAbs. After two days, vehicle or Xe-C was added to the cells and IL-2 and IFNγ production was examined by ELISA two days later. (E) CD4 T cells were activated with anti-CD3/anti-CD28 mAbs in the presence (AP) or absence (−) of 2-APB and RNA was examined 24h later by RPA. L32 and GAPDH were used as loading controls. (F) CD4 T cells were activated with anti-CD3/anti-CD28 mAbs. Medium alone (-) or 2-APB (AP) was added on day 2 and RNA was analyzed by RPA on day 3. L32 and GAPDH were used as loading controls. (G) CD4 T cells were activated with anti-CD3 and anti-CD28 mAbs and after 6, 12 or 24h Xe-C or vehicle was added to the culture. IL-2 and IFNγ production were examined by ELISA at 48h from initial activation. (H) CD4 T cells were activated in the presence of IL-2 (20 ng/ml) or medium alone. Xe-C or vehicle was added at 24h of activation and supernatants were analyzed for IFNγ production at 48h